The role of the transcription factor Brachyury (Bra) in notochord formation is well documented and evolutionarily conserved throughout the chordate phylum. However, still little is known about the transcription factors controlled directly or indirectly by Bra and about the function of factors acting in concert with Bra in notochord development and evolution. In particular, the knowledge of structure, common features and modalities of regulation of cis-regulatory modules (enhancers) directing expression in the notochord is fragmentary and poor. We have exploited the experimental advantages offered by the ascidian Ciona intestinalis as a model system to isolate and characterize a collection of notochord enhancers from genes that have already been described as Bra targets and from genes whose relationship with Bra is unknown. Our preliminary data, gathered from the comparative analysis of the notochord enhancers, lead us to hypothesize that notochord formation in Ciona relies upon additional, mostly uncharacterized notochord transcription factors that fall into two categories: Bra-downstream factors, controlled directly or indirectly by Bra and acting as its transcriptional intermediaries and factors acting in concert with Bra. We will test these hypotheses through the following specific aims: (1) Isolate minimal regulatory sequences necessary for notochord expression;(2) Identify the activators controlling the minimal enhancer sequences and study their role in notochord development;(3) Identify the enhancers controlling, in turn, selected notochord activators. The broad scope of the proposed research is to rapidly untangle the gene networks underlying the different steps of notochord development and differentiation and to gain valuable insights into the molecular mechanisms controlling gene expression in the notochord during its formation and evolution.